Water for DNA Sequencing
Impact of Water
DNA sequencing can be viewed as a combination of three techniques or steps:
- Step 1: The generation of DNA fragments, generally by a PCR-based technique
- Step 2: The separation of the fragments by capillary or regular agarose gel electrophoresis
- Step 3: The detection step, most frequently fluorescence detection.
A brief outlook of the impact of the water quality is provided here. Water quality used to operate the DNA sequencer should meet some quality criteria in order to optimize the sequencing process.
Nuclease-free water
Nuclease-free water is recommended to avoid the degradation of the DNA at all steps of the DNA sequencing. Nuclease removal is efficiently done using ultrafiltration devices (see the section on RNase-free water). Point-of use ultrafiltration cartridges can be installed at the outlet of water purification systems to provide nuclease-free water on demand.
Organics
Organics are the most disruptive contaminants in DNA sequencing. Indeed, high load of organics will impair the gel electrophoresis process, inside the capillary or in larger gels. Large organic acids, such as humic and fulvic acids resulting from natural matter degradation can co-elute with the DNA fragments during the electrophoresis, and can disturb the polymerase efficiency as well during the PCR step. In addition, and more frequently, some organics can interfere with the fluorescence detection and contribute to errors in the sequencing of the target DNA analyzed. Most sequencing techniques, indeed, are based on the detection of fluorescent dyes attached to the DNA fragments. The presence of fluorescence absorbing and quenching organic molecules can disturb the detection.
In order to ensure organic purity of the high purity water utilized for DNA sequencing, a low overall organic content, referred to as Total Organic Carbon (TOC) is recommended. Specifications of TOC < 5 ppb (µg/L) are achievable on high purity water purification systems.
Ions
The levels of some ions, such as magnesium, need to be carefully adjusted during the PCR step, in order to optimize the efficiency of the polymerase. Other ions, cadmium and some other divalent cations, must be removed to prevent inhibition of the polymerase. The ionic force needed during the electrophoresis process is also best obtained and controlled when the water used ahs a high purity, and does not contain unknown concentrations of ions.
High resistivity (18.2 MΩ•cm) ensures low ionic content (below 1 µg/L of overall ionic concentration) in the water. High purity water purification systems are designed to provide continuously high resistivity water on demand.
Bacteria
Bacteria can release nucleases in the water, and therefore, should be removed to avoid degradation of the nucleic acid. Bacteria also release DNA that could interfere with the electrophoresis separation of the target DNA fragments. In addition, bacteria release ions and organics, both contaminants that can interfere with the DNA sequencing, as mentioned previously.
The control of bacteria level with water purification systems is obtained via a combination of technologies and maintenance recommendation throughout the purification process. Membrane technologies, absolute 0.2 µm microfiltration or ultrafiltration, can remove bacteria at the point of delivery and ensure bacteria levels < 0.1 cfu/mL. Germicidal UV at 254 nm.
Overall, during the sequencing step, the water quality should meet the following criteria:
- nuclease-free
- high resistivity (18.2 MΩ•cm)
- low organic content (TOC < 5 ppb)
- low bacteria count (< 1 cfu/mL)
More Information
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